To adapt to harsh environmental stresses such as extremes of temperature, dehydration, and high osmotic pressure, certain organisms have accumulated intracellular concentrations of solutes called osmolytes . Osmolytes protect proteins and other cell components against the extreme conditions and do so without significantly altering the biological activity of the macromolecule. These characteristics functionally describe these osmolytes as compatible. In adaptations in which urea is concentrated in cells, as in sharks and in mammalian kidney, methylamine osmolytes are concentrated intracellulary to protect cell proteins against the deleterious effects of urea. These osmolytes are said to be counteracting and are believed to alter the biological activity of proteins in a direction opposite to that of urea. The long- term goals of our research are two fold: (I) to understand the mechanisms by which natural occurring osmolytes protect proteins against deleterious structural effects brought about by stresses of heat, cold, high salt, and urea normally encountered in living systems, and (II) to understand the mechanisms by which compatible and counteracting osmolytes ensure appropriate functional activity of proteins in the fact of these stresses. A major focus will be on osmolytes of mammalian kidney, including the compatible osmolytes sorbitol and inositol, and the counteracting osmolytes glycerolphosphoryl choline (GCP) and betaine. We will use transfer free energy measurements of amino acid side chains and the peptide backbone from water to proline, valine, arginine, sorbitol, inositol, GPC, and betaine, and to GPC/urea and sorbitol/urea mixtures. These data will be used to evaluate the transfer free energies of native and fully unfolded forms of RNase A from water to these solute-containing solutions. The objective is to determine which functional groups on the protein are responsible for protein stabilization, and in the case of arginine and valine as solutes, to determine which are responsible for protein destabilization. Such information is essential for understanding the molecular bases for protein stabilization by osmolytes. The effects of these solutes on NMR-detected hydrogen exchange (HX) kinetics of RNase A amide protons will be determined for the purpose of investigating the molecular basis of compatibility and counteraction. In the case of urea affects on protein structural integrity, HX will be used to determine how kidney osmolytes offset these effects.